Toxoplasma gondii is an intracellular protozoan parasite of warm-blooded animals, and has infected up to one-third of the human population. The replicative stage (tachyzoite) develops into a latent stage (bradyzoite) that resides inside host cells as cysts in brain, heart, and skeletal muscle tissues. These tissue cysts persist within the host for life, as they are impervious to the immune response and currently approved anti-parasitic drugs (e.g. antifolates). Tissue cysts give rise to recurrent reactivation of infection in immune compromised patients, and some studies have correlated latent toxoplasmosis with various neurological disorders, including schizophrenia. One of the most pressing needs in the field is the discovery of chemical entities possessing the ability to attack and reduce tissue cysts. In recent studies, we demonstrated that guanabenz (GA), an old drug used to treat hypertension, also has potent anti-parasitic activity against Toxoplasma through its ability to interfere with parasite translational control. GA is already FDA-approved and can cross the blood-brain barrier, making it an attractive candidate for repurposing as a drug to treat toxoplasmosis. In a mouse model of infection, we showed that GA displayed modest activity against acute infection, but remarkable activity against bradyzoite tissue cysts in latent toxoplasmosis, reducing the brain cyst burden 70-80% compared to vehicle controls. GA thus represents one of the first drugs that demonstrates it is possible to reduce tissue cyst levels in infected animals. In unrelated studies, our collaborator, Dr. Stone Doggett, found that endochin-like quinolones (ELQs), which target the parasite's cytochrome bc1 complex, can reduce brain cyst burden up to ~85%. Intriguingly, these two drugs, which have differing mechanisms of action, each lacks the ability to fully eradicate cysts. We hypothesize that these remaining cysts arise from residual tachyzoites that can be nullified through the use of a drug combination. In this R21 application, we propose to address this hypothesis with two specific aims: (i) after treating latently infected mice with GA or ELQ, we will examine specific brain regions to determine where the intractable cysts remain, and determine whether other organs harbor parasites that evade drug treatment; (ii) we will determine whether novel combinations of GA and ELQ, or co-administration of antifolates, will reduce tissue cysts to undetectable levels. The identification of a pharmacological strategy that reduces or eliminates Toxoplasma tissue cysts would be a significant advance towards a radical cure for toxoplasmosis.